Aquatic birnavirus induces necrotic cell death via the mitochondria-mediated caspase pathway

Aquatic birnavirus induces necrotic cell death by an ill-understood process. Presently, we demonstrate that infectious pancreatic necrosis virus (IPNV) induces post-apoptotic necrotic cell death through loss of mitochondrial membrane potential (MMP) followed by caspase-3 activation in CHSE-214 cells. Progressive phosphatidylserine externalization was observed at 6 h post-infection (p.i.). This was followed by the development of bulb-like vesicles (bleb formation) at 8 h p.i. Progressive loss of MMP was also observed in IPNV-infected CHSE-214 cells beginning at 6 h p.i. At 8 h and 12 h p.i., IPNV-infected cells demonstrated a dramatic increase in MMP loss, rapid entry into necrotic cell death, and activation of caspase-9 and -3. Additionally, treatment with an inhibitor of MMP loss, bongkrekic acid, an adenine nucleotide translocase inhibitor, blocked IPNV-induced PS exposure and MMP loss, as well as reduced the activation of caspase-3. Taken together, our results suggest that IPNV induces apoptotic cell death via loss of MMP, thereby triggering secondary necrosis and caspases-3 activation. Furthermore, this death-signaling pathway is disrupted by bongkrekic acid in fish cells, indicating that this drug may serve to modulate IPNV-induced pathogenesis.     

Lymphoid apoptosis in Edwardsiella tarda septicemia in tilapia, Oreochromis niloticus

The present study revealed a relationship between the kinetic change of apoptosis and the inflammatory response during experimental intraperitoneal infection with Edwardsiella tarda as a septicemic model. The morphological changes of apoptotic cells including cellular shrinkage, condensed nuclear chromatin, nuclear fragmentation and membrane blebbing were detected by light and transmission electron microscopy. TUNEL and agarose gel electrophoresis confirmed the fragmentation of DNA in the apoptotic cells. Apoptosis was highly detected in lymphoid organs prior to the inflammatory process and gradually decreased after an extensive inflammatory response. Apoptosis in thymus and spleen was extensive and an in vitro study revealed that lymphocytes were the major cell population which underwent apoptosis. The result suggests that E. tarda-induced systemic immunosuppression via lymphocyte apoptosis as determined by suppression of the systemic inflammatory response during an initial step of generalized septicemia.     

Blebs produced by actin–myosin contraction during apoptosis release damage-associated molecular pattern proteins before secondary necrosis occurs

Apoptosis is a fundamental homeostatic mechanism essential for the normal growth, development and maintenance of every tissue and organ. Dying cells have been defined as apoptotic by distinguishing features, including cell contraction, nuclear fragmentation, blebbing, apoptotic body formation and maintenance of intact cellular membranes to prevent massive protein release and consequent inflammation. We now show that during early apoptosis limited membrane permeabilization occurs in blebs and apoptotic bodies, which allows release of proteins that may affect the proximal microenvironment before the catastrophic loss of membrane integrity during secondary necrosis. Blebbing, apoptotic body formation and protein release during early apoptosis are dependent on ROCK and myosin ATPase activity to drive actomyosin contraction. We identified 231 proteins released from actomyosin contraction-dependent blebs and apoptotic bodies by adapted SILAC (stable isotope labeling with amino acids in cell culture) combined with mass spectrometry analysis. The most enriched proteins released were the nucleosomal histones, which have previously been identified as damage-associated molecular pattern proteins (DAMPs) that can initiate sterile inflammatory responses. These results indicate that limited membrane permeabilization occurs in blebs and apoptotic bodies before secondary necrosis, leading to acute and localized release of immunomodulatory proteins during the early phase of active apoptotic membrane blebbing. Therefore, the shift from apoptosis to secondary necrosis is more graded than a simple binary switch, with the membrane permeabilization of apoptotic bodies and consequent limited release of DAMPs contributing to the transition between these states.     

Frequent occurrence of apoptosis is not associated with pathogenic infectious pancreatic necrosis virus (IPNV) during persistent infection

Infectious pancreatic necrosis virus (IPNV), a member of the genus Aquabirnavirus and family Birnaviridae, is an unenveloped icosahedral virus with two segments of double-stranded RNA. IPNV causes acute infection in salmonid fry and fingerlings with high mortality. However, this mortality is low as the age increases and survivors become IPNV-carrier fish. In this study, IPNV persistent infection was established in rainbow trout with no clinical signs or mortality. TUNEL staining and immunohistochemistry showed that IPNV antigen-positive cells did not have an apoptotic nucleus in almost all tissue sections and leucocyte smears, indicating that apoptosis was not induced in IPNV antigen-positive cells. The IPNV genome detected by in situ RT-PCR was more frequent than detection of the IPNV antigen by immunohistochemistry in the kidney, spleen, and liver. This result implies that the successive replication would not occur in many IPNV-infected cells. Further, apoptotic cells were predominant in the tissue sections where the signal-positive cells were frequently detected. Therefore, the presence of apoptosis in this study might be associated with host defense mechanisms, which eliminates IPNV-infected cells by the recognition of IPNV genome at the early stage of infection.     

Aquatic birnavirus-induced ER stress-mediated death signaling contribute to downregulation of Bcl-2 family proteins in salmon embryo cells

Aquatic birnavirus induces mitochondria-mediated cell death, but whether connects to endoplasmic reticulum (ER) stress is still unknown. In this present, we characterized that IPNV infection triggers ER stress-mediated cell death via PKR/eIF2α phosphorylation signaling for regulating the Bcl-2 family protein expression in fish cells. The IPNV infection can induce ER stress as follows: (1) ER stress sensor ATF6 cleavaged; (2) ER stress marker GRP78 upregulation, and (3) PERK/eIF2α phosphorylation. Then, the IPNV-induced ER stress signals can induce the CHOP expression at early (6 h p.i.) and middle replication (12 h p.i.) stages. Moreover, IPNV-induced CHOP upregulation dramatically correlates to apparently downregulate the Bcl-2 family proteins, Bcl-2, Mcl-1 and Bcl-xL at middle replication stage (12 h p.i.) and produces mitochondria membrane potential (MMP) loss and cell death. Furthermore, with GRP78 synthesis inhibitor momitoxin (VT) and PKR inhibitor 2-aminopurine (2-AP) treatment for blocking GRP78 expression and eIF2α phosphorylation, PKR/PERK may involve in eIF2α phosphorylation/CHOP upregulation pathway that enhances the downstream regulators Bcl-2 family proteins expression and increased cell survival. Taken together, our results suggest that IPNV infection activates PKR/PERK/eIF2α ER stress signals for regulating downstream molecules CHOP upregulation and Bcl-2 family downregulation that led to induce mitochondria-mediated cell death in fish cells, which may provide new insight into RNA virus pathogenesis and disease.     

Stage-specific expression of TNFα regulates bad/bid-mediated apoptosis and RIP1/ROS-mediated secondary necrosis in Birnavirus-infected fish cells

Infectious pancreatic necrosis virus (IPNV) can induce Bad-mediated apoptosis followed by secondary necrosis in fish cells, but it is not known how these two types of cell death are regulated by IPNV. We found that IPNV infection can regulate Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic death pathways via the up-regulation of TNFα in zebrafish ZF4 cells. Using a DNA microarray and quantitative RT-PCR analyses, two major subsets of differentially expressed genes were characterized, including the innate immune response gene TNFα and the pro-apoptotic genes Bad and Bid. In the early replication stage (0-6 h post-infection, or p.i.), we observed that the pro-inflammatory cytokine TNFα underwent a rapid six-fold induction. Then, during the early-middle replication stages (6-12 h p.i.), TNFα level was eight-fold induction and the pro-apoptotic Bcl-2 family members Bad and Bid were up-regulated. Furthermore, specific inhibitors of TNFα expression (AG-126 or TNFα-specific siRNA) were used to block apoptotic and necrotic death signaling during the early or early-middle stages of IPNV infection. Inhibition of TNFα expression dramatically reduced the Bad/Bid-mediated apoptotic and Rip1/ROS-mediated necrotic cell death pathways and rescued host cell viability. Moreover, we used Rip1-specific inhibitors (Nec-1 and Rip1-specific siRNA) to block Rip1 expression. The Rip1/ROS-mediated secondary necrotic pathway appeared to be reduced in IPNV-infected fish cells during the middle-late stage of infection (12-18 h p.i.). Taken together, our results indicate that IPNV triggers two death pathways via up-stream induction of the pro-inflammatory cytokine TNFα, and these results may provide new insights into the pathogenesis of RNA viruses.     

Use of the monoclonal antibody DAKO-ERbeta (8D5-1) to measure oestrogen receptor beta in breast cancer cells

BACKGROUND: Following a lethal injury, two modes of cell death can be distinguished, apoptosis and primary necrosis. Cells pass through a prelethal stage characterized by a preservation of membrane integrity, in which they shrink (apoptosis) or swell (oncosis, the early phase of primary necrosis). During apoptosis, a loss of phospholipid asymmetry leads to exposure of phosphatidylserine (PS) residues on the outer leaflet of the plasma membrane. We examined whether the external PS exposure, initially supposed to be specific for apoptosis, was also observed in oncotic cells. METHODS: Human peripheral lymphocytes, Jurkat T cells, U937 cells, or HeLa cells were submitted to either apoptotic or oncotic stimuli. PS external exposure was assessed after binding of FITC-conjugated annexin V as was the loss of membrane integrity after propidium iodide (PI) uptake. Morphological examination was performed by optical or electron microscopy. RESULTS: Similarly to apoptotic cells, oncotic cells expose external PS residues while preserving membrane integrity. Consequently, oncotic cells exhibit the annexin V+ PI- phenotype, previously considered to be specific for apoptotic cells. CONCLUSIONS: This study concludes that the annexin V/PI assay does not discriminate between apoptosis and oncosis and that it can be a useful tool to study oncosis by flow cytometry.     

Phagocytosis of necrotic cells by macrophages is phosphatidylserine dependent and does not induce inflammatory cytokine production

Apoptotic cells are cleared by phagocytosis during development, homeostasis, and pathology. However, it is still unclear how necrotic cells are removed. We compared the phagocytic uptake by macrophages of variants of L929sA murine fibrosarcoma cells induced to die by tumor necrosis factor-induced necrosis or by Fas-mediated apoptosis. We show that apoptotic and necrotic cells are recognized and phagocytosed by macrophages, whereas living cells are not. In both cases, phagocytosis occurred through a phosphatidylserine-dependent mechanism, suggesting that externalization of phosphatidylserine is a general trigger for clearance by macrophages. However, uptake of apoptotic cells was more efficient both quantitatively and kinetically than phagocytosis of necrotic cells. Electron microscopy showed clear morphological differences in the mechanisms used by macrophages to engulf necrotic and apoptotic cells. Apoptotic cells were taken up as condensed membrane-bound particles of various sizes rather than as whole cells, whereas necrotic cells were internalized only as small cellular particles after loss of membrane integrity. Uptake of neither apoptotic nor necrotic L929 cells by macrophages modulated the expression of proinflammatory cytokines by the phagocytes.     

Changes in elemental content during apoptotic cell death studied by electron probe X-ray microanalysis

Recent data suggest that changes in ionic content, primarily potassium, play a pivotal role in the progression of apoptosis. However, the changes in total element content, i.e., sodium (Na), magnesium (Mg), phosphorous (P), chlorine (Cl), potassium (K), and calcium (Ca), during apoptosis have not been evaluated. Electron probe X-ray microanalysis (EPXMA) was used to measure total element content in U937 cells before and after the induction of apoptosis. As an experimental model we used U937 cells irradiated with ultraviolet (UV) light. Apoptosis was evaluated with phase-contrast microscopy, with scanning and transmission electron microscopy, and with the fluorescent dye bisbenzimide (Hoechst 33342). Plasma membrane permeability as a measure of cell death was determined by trypan blue dye exclusion. To investigate element content with EPXMA, cells were cryoprepared, i.e., cryofixed and freeze-dried, and analyzed as whole cells using a scanning electron microscope. We found that the UV irradiation induced rapid (within 2 h) morphological changes associated with apoptosis, such as plasma membrane blebbing, condensation of the chromatin, and the formation of membrane-bound apoptotic bodies. At this time, 95% of the apoptotic cells excluded trypan blue dye. EPXMA results demonstrated that UV light-irradiated apoptotic cells (cells with membrane-bound apoptotic bodies) had a lower Cl content (P < 0.001) and K content (P < 0.001) and a higher Na content (P < 0.001) in comparison with nonirradiated control cells. Also, P and Ca content was higher in apoptotic cells than in control cells, but this difference did not reach statistical significance. No differences were found in Mg. These data indicated that morphological changes characteristic of apoptotic cell death are related with significant changes in sodium, chlorine, and potassium content. In addition, we demonstrated that these changes in elemental composition were not associated with loss of cell membrane integrity.     

Computerized video time-lapse microscopy studies of ionizing radiation-induced rapid-interphase and mitosis-related apoptosis in lymphoid cells

Computerized video time-lapse (CVTL) microscopy of X-irradiated cultures of cells of the murine lymphoma cell lines ST4 and L5178Y-S and the human lymphoid cell line MOLT-4 demonstrated that these cells exhibit a wide disparity in the timing of induction and execution of radiation-induced cell death that included rapid-interphase apoptosis, delayed apoptosis, and postmitotic apoptosis. ST4 cells that received 2.5 or 4 Gy of X radiation underwent rapid-interphase apoptosis within 2 h. Apoptosis commenced with a 10-20-min burst of membrane blebbing followed by swelling for 2-4 h and cell collapse. No apoptotic bodies were formed. After a dose of 1 Gy, approximately 90% of ST4 cells died by rapid-interphase apoptosis, while the remainder completed several rounds of cell division prior to cell death. Postmitotic death of ST4 cells occurred with the same morphological sequence of events as during rapid-interphase apoptosis induced by doses of 1-4 Gy. In contrast, L5178Y-S and MOLT-4 cells that received 4 Gy underwent apoptosis more slowly, with a complex series of events occurring over 30-60 h. Only 3% of L5178Y-S cells and 24% of MOLT-4 cells underwent apoptosis without attempting cell division. The cells became abnormally large during a long G(2)-phase delay, and then most of the cells (76-97%) attempted to divide for the first or second time at approximately 18-30 h postirradiation. However, either mitosis failed or division was aberrant; i.e., the large cells divided into three or four fragments which eventually fused together. This process was followed by several rounds of complex and unpredictable membrane blebbing, gross distortions of shape, fragmentation-refusion events, and formation of apoptotic bodies, after which the cells collapsed at 36-60 h postirradiation.     

Zebrafish anti-apoptotic gene Bcl-xL can prevent aquatic birnavirus-induced cell death in fish cells without affecting expression of viral proteins

The aquatic birnavirus induces mitochondria-mediated cell death in fish; however, the molecular mechanism remains unknown. In the present study, we demonstrated that aquatic birnavirus-induced mitochondria-mediated cell death is regulated by the anti-apoptotic Bcl-2 family member, zfBcl-xL, which is anti-apoptotic and enhances host cell viability. First, CHSE-214 cells carrying EGFP-zfBcl-xL fused genes were selected, established in culture, and used to examine the involvement of zfBcl-xL in host cell protection from the effects of viral infection. EGFP-zfBcl-xL was found to prevent infectious pancreatic necrosis virus (IPNV)-induced phosphatidylserine exposure up to 40% at 12 h and 24 h post-infection (p.i.), block IPNV-induced loss of mitochondrial membrane potential (ΔΨm), and enhance host viability at the middle and late replication stages. In addition, zfBcl-xL overexpression prevented IPNV-induced caspase-9 activation up to 25% and 85% at the middle (12 h p.i.) and late (24 h p.i.) replication stages without affecting expression of viral proteins such as VP3 (as a viral death protein) protein. In the present study, we demonstrated that aquatic birnavirus-induced cell death is prevented by the anti-apoptotic Bcl-2 family member, zfBcl-xL, which enhances host cell viability through blockage of mitochondrial disruption and caspase-9 activation.     

Process of apoptosis induced by TNF-α in murine fibroblast Ltk-cells: Continuous observation with video enhanced contrast microscopy

Apoptosis is originally defined by unique morphological changes of dying cells, and the biochemical hallmark associated with apoptosis is internucleosomal DNA fragmentation. However, few report has shown the precise time course of the apoptotic events. The present study was designed to try to clarify apoptotic processes using a video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy. The morphological changes of murine fibroblast Ltk-cells treated with TNF- were divided into four stages: (i) pre-apoptotic, (ii) cytoplasmic shrinkage, (iii) membrane blebbing, and (iv) ballooning. Almost of the cells underwent cytoplasmic shrinkage and membrane blebbing within 6 hours after TNF- exposure, and at about 9 hours, they were in the ballooning stage. Based on these data, we investigated the relationship between morphological changes and other biochemical features. The earliest event was exposure of phosphatidyl-serine at the cytoplasmic membrane, which was already observed in the pre-apoptotic stage. Loss of mitochondrial membrane potential was observed in the cytoplasmic shrinkage stage. Caspase-8/-3 activities already started increasing in the pre-apoptotic stage, and reached their peak at 6 hours after TNF- exposure. DNA fragmentation occurred in the late phase of the membrane blebbing.     

Apoptotic Membrane Blebbing Is Regulated by Myosin Light Chain Phosphorylation

The evolutionarily conserved execution phase of apoptosis is defined by characteristic changes occurring during the final stages of death; specifically cell shrinkage, dynamic membrane blebbing, condensation of chromatin, and DNA fragmentation. Mechanisms underlying these hallmark features of apoptosis have previously been elusive, largely because the execution phase is a rapid event whose onset is asynchronous across a population of cells. In the present study, a model system is described for using the caspase inhibitor, z-VAD-FMK, to block apoptosis and generate a synchronous population of cells actively extruding and retracting membrane blebs. This model system allowed us to determine signaling mechanisms underlying this characteristic feature of apoptosis. A screen of kinase inhibitors performed on synchronized blebbing cells indicated that only myosin light chain kinase (MLCK) inhibitors decreased blebbing. Immunoprecipitation of myosin II demonstrated that myosin regulatory light chain (MLC) phosphorylation was increased in blebbing cells and that MLC phosphorylation was prevented by inhibitors of MLCK. MLC phosphorylation is also mediated by the small G protein, Rho. C3 transferase inhibited apoptotic membrane blebbing, supporting a role for a Rho family member in this process. Finally, blebbing was also inhibited by disruption of the actin cytoskeleton. Based on these results, a working model is proposed for how actin/myosin II interactions cause cell contraction and membrane blebbing. Our results provide the first evidence that MLC phosphorylation is critical for apoptotic membrane blebbing and also implicate Rho signaling in these active morphological changes. The model system described here should facilitate future studies of MLCK, Rho, and other signal transduction pathways activated during the execution phase of apoptosis.     

Live fluorescence and transmission-through-dye microscopic study of actinomycin D-induced apoptosis and apoptotic volume decrease

The effect of actinomycin D on HeLa cells was studied by live fluorescence and transmission-through-dye microscopy—a recently developed technique that permits volume measurements in live cells. In particular, it is well suited for the observation and quantification of the apoptotic volume decrease (AVD), which is widely viewed as an essential feature of apoptosis. The main results from our study are as follows. (1) Apoptosis caused in HeLa cells by actinomycin D proceeds in two morphologically distinct stages: the early stage is characterized by extensive blebbing, and the late stage by a more compact shape. The loss of mitochondrial membrane potential occurs at about the same time as blebbing, and chromatin condensation follows 30–90 min later. Caspase-3 and 7 become activated during the late stage. (2) Because blebbing occurs before activation of caspase-3, it has to be initiated by a different mechanism. Although blebbing is one of the earliest observable changes, it can be selectively inhibited without affecting other apoptotic reactions. (3) The majority of cells experience a temporary volume increase after the appearance of blebs. Eventually, AVD takes over and the cells shrink by approximately 40 % of their initial volume; the volume loss becomes noticeable at the end of the blebbing phase and continues through the late stage. Sometimes, at the end of long incubations, shrinkage gives way to swelling, possibly indicating secondary necrosis. (4) Both early and late apoptosis are accompanied by intracellular accumulation of Na⁺, while low-sodium medium prevents apoptosis. Except for a partial protective effect of quinine, all of the tested blockers of Na⁺, K⁺ and Cl^? channels failed to prevent apoptosis or AVD.     

Synchronized onset of nuclear and cell surface modifications in U937 cells during apoptosis

In this study we investigated the relationship between nuclear and cell surface modifications (i.e. blebbing, phosphatidylserine [PS] and sugar residues exposure) in a monocytic cell line, U937, during apoptosis induced by oxidative stress (1 mM H2O2) or inhibition of protein synthesis (10 microg/ml puromycin). Dying cells were simultaneously observed for nuclear modifications, presence of superficial blebs and plasma membrane alterations. Morphological analysis performed by conventional fluorescence microscopy, or by transmission and scanning electron microscopy showed that the courses of nuclear and membrane alterations occured concomitantly, but the phenotype was dependent on the stage of the apoptotic process and the type of apoptogenic inducer used. The progression of apoptosis in U937 cells beyond early stages resulted in the extensive formation of blebs which concomitantly lost some typical markers of apoptosis, such as PS and sugar residues. Therefore, the modality by which the nucleus condenses, or the amount and the pattern of distribution of PS on the cell surface were, for each cell line, strictly related to the apoptogenic inducer. The morphological data reported in the present paper should lead to a more precise quantification of apoptosis by improving the detection of apoptotic cells in vivo (i.e. in tissue, organs), which is a crucial point in the evaluation of efficiency of antiproliferative drugs, such as antiblastic or immunosuppressive compounds.     

Proteolytic specificity of caspases is required to signal the appearance of apoptotic morphology

The caspase family of cysteine proteases is essential for implementation of physiological cell death. Since a wide variety of cellular proteins is cleaved by caspases during apoptosis, it has been predicted that digestion of proteins crucial to maintaining the life of a cell is central to apoptosis. To assess the role of the proteolytic destruction during apoptosis, we introduced the non-specific protease proteinase K into intact cells. This introduction led to extensive digestion of cellular proteins, including physiological caspase-substrates. Caspase-3-like activity was induced rapidly, followed by morphological signs of apoptosis such as membrane blebbing and nuclear condensation. The caspase inhibitor Z-VAD-fmk inhibited the appearance of these morphological changes without reducing the extent of intracellular proteolysis by proteinase K. Loss of integrity of the cell membrane, however, was not blocked by Z-VAD-fmk. This system thus generated conditions of extensive destruction of caspase substrates by proteinase K in the absence of apoptotic morphology. Taken together, these experiments suggest that caspases implement cell death not by protein destruction but by proteolytic activation of specific downstream effector molecules.     

Assessment of apoptosis in xenobiotic-induced immunotoxicity.

Generation of immunity is a highly complex process in which proliferation and differentiation of immune-competent cells regulated by cytokines and cell-cell interactions play a major role. Reducing the number of immune-competent cells or altering the function, selection, and differentiation of lymphocytes after xenobiotic treatment may lead to serious adverse effects. Programmed cell death, or apoptosis, is a highly regulated process by which an organism eliminates unwanted cells without eliciting an inflammatory response. However, xenobiotics are also able to trigger unwanted apoptosis or to alter the regulation of programmed cell death. Cytological characteristics of apoptosis are generally different from those seen in acute pathological cell death resulting from cell injury. The morphological characteristics of apoptosis are unique including cell shrinkage, membrane blebbing, chromatin condensation, DNA fragmentation, disruption of the nuclear lamina, nuclear fragmentation, and emergence of apoptotic bodies. It is now established that apoptosis plays a critical role in both development and homeostasis of the immune system: thymic selection, cytotoxicity, deletion of autoreactive cells, and regulation of the size of the lymphoid compartment. Assessment of apoptosis relies on the morphological and biochemical modifications of the dying cells. As a rule, and because an apoptotic cell rarely displays all of the characteristic apoptotic features, several criteria should be monitored in parallel including morphological examination. The techniques described in this paper have been divided into five categories: analysis of cell morphology by microscopy, identification of DNA fragmentation, determination of mitochondrial membrane potential, detection of plasma membrane changes, analysis of caspase activation.     

U937 variant cells as a model of apoptosis without cell disintegration

The variant cell line U937_V was originally identified by a higher sensitivity to the cytocidal action of tumor necrosis factor alpha (TNFα) than that of its reference cell line, U937. We noticed that a typical morphological feature of dying U937_V cells was the lack of cellular disintegration, which contrasts to the formation of apoptotic bodies seen with dying U937 cells. We found that both TNFα, which induces the extrinsic apoptotic pathway, and etoposide (VP-16), which induces the intrinsic apoptotic pathway, stimulated U937_V cell death without cell disintegration. In spite of the distinct morphological differences between the U937 and U937_V cells, the basic molecular events of apoptosis, such as internucleosomal DNA degradation, phosphatidylserine exposure on the outer leaflet of the plasma membrane, caspase activation and cytochrome c release, were evident in both cell types when stimulated with both types of apoptosis inducer. In the U937_V cells, we noted an accelerated release of cytochrome c, an accelerated decrease in mitochondrial membrane potential, and a more pronounced generation of reactive oxygen species compared to the reference cells. We propose that the U937 and U937_V cell lines could serve as excellent comparison models for studies on the mechanisms regulating the processes of cellular disintegration during apoptosis, such as blebbing (zeiosis) and apoptotic body formation.     

Culture filtrates of Aspergillus fumigatus induce different modes of cell death in human cancer cell lines

Aspergillus fumigatus culture filtrate (CF) has a potent cytotoxic effect on three human cancer cell lines (DLKP, A549 and HEp-2) and initiates cell death by apoptosis but the execution of the apoptotic process is incomplete. DLKP cells treated with A. fumigatus CF demonstrate features associated with apoptosis but cytoplasmic and nuclear fragmentation were not observed and cells ultimately underwent necrosis. The apoptotic process commenced in A549 and HEp-2 cells upon exposure to CF, cell shrinkage was observed but membrane blebbing and apoptotic body formation were not detected and detached cells died by necrosis. In contrast, extensive nuclear fragmentation and apoptotic body formation were evident in DLKP and A549 cells treated with anti-neoplastic agents. This work indicates that A. fumigatus CF is cytotoxic to cancer cells and can initiate apoptosis but that the complete apoptotic pathway is not followed. This revised version was published online in June 2006 with corrections to the Cover Date.